For many confocal, two-photon and TDI scanning microscopes, a very fast autofocus procedure is required. This fast procedure needs to function properly in combination with large imaging objective numerical apertures (>0.5) and with samples material that is located underneath a reflective surface, for instance, sliced tissue as used in histology.
In the existing technologies, several approaches for auto-focusing are known. The majority of these are based on special frequency analysis of an image taken from the sample by use of an imaging camera. This approach is limited with respect to response speed, as they are based on iterative optimization to set the focus into the sample. In the applications of well plate inspection and large area slide scanning using TDI (time delay integration) imaging, very short response times of the autofocus device are demanded. The technique of special frequency analysis is the limiting factor in optimizing the speed of these imaging problems.
Alternative technologies, like triangulation or patterned illumination analysis, use a much simpler and faster approach. The drawback of these is that they are highly sensitive to signal disturbance that is caused by reflective surfaces located close to the sample to be imaged.
Using low coherence interferometry for determining the location of a sample, leading to a focus adjustment, has been proposed earlier (U.S. Pat. No. 5,493,109). The proposed fashion is not sufficient to solve the problems of high precision (focusing repeatability <1 μm in the optical axis) and reflection sensitivity (when imaging through a glass surface). The reason for these problems is that the proposed setup is utilizing a Michelson type interferometer where the interferometer arms are significantly separated locally and partially guided through a fiber. This approach is subject to thermally and mechanically induced changes to the optical path length of one interferometer arm with respect to the other. Holding a repeatability of <1 μm in the proposed system has shown to be impossible. The target application of this is for surgical ophthalmic OCT (optical coherence interferometry) that does not require such large repeatability, as lower NAs (<0.5) are used in these. Another description of a related technology is found in WO2012016753A1. The proposed approach comes closer to solving the requirements that has been seen in the above mentioned target applications, as it proposes a beam splitter located close to the imaging objective and hence reduces the relative optical path length drift in between the interferometer arms (see claim 5). The product and procedure described includes no means of suppressing reflexes from surfaces located close to the sample and is hence not suitable for high NA (>0.5) imaging of samples covered by glass. In these situations, the large signals induced by glass reflexes can overlap with signal from the sample, so that absolute definition of the sample position with <1 μm accuracy is not possible.
FIG. 1 shows a low coherence interferometry signal derived from a microscopic setup using a Gaussian beam that is centered on the optical axis of an imaging microscope with a slide that holds a thin (˜5 μm thick) histological sample. The sample is covered by a glass cover slip. The following features are visible:
Auto-interference (A) caused by spectral fringes created through interference from mainly the first surface of the cover slip (B) and adjacent reflexes from second surface of the cover slip and the slide glass (C). The back surface of the cover slip creates another signal peak (D).
The signal created by the sample is expected between the two peaks around position (C) and is superimposed by glass reflexes. A precise (<1 μm) determination of the sample position is not possible given such data set.
Therefore, there is a need for products and procedures that can solve the above problems.
An embodiment of the present invention proposes procedures for setting the illumination and detection aperture by ways of displacement from the optical axis while staying parallel to this axis. This is in principal different from the procedure suggested by Zeiss AG in WO2012016753A1, where an angled beam before the objective is used to create a parallel offset of the measurement beam with respect to the center optical axis. This approach is not capable of suppressing surface reflexes well enough to allow precise definition of sample location within <1 μm axial accuracy in thin (<10 μm thickness) samples that are placed underneath a microscope cover slip.
Furthermore, none of these two low coherence interferometry approaches discussed above suggest a solution for optimizing the signal when an imaging objective is changed.
An embodiment of the present invention provides a procedure that numerically compensates for offsets in the control loop that derive from use of arbitrary thickness material that is located in between imaging objective and sample material. No existing literature was found describing the compensation of refraction induced by material covering the sample.
Some embodiments of the present invention provide procedures for automatically calibrating the device for use of different imaging objectives. No existing literature was found describing such automatic calibration.